168
J. M e d . Chem. 1991,34, 168-174
mL of cold buffer. The radioactivity associated with the filter was determined by liquid-scintillation spectrometry. Nonspecific binding waa defined with 60 pM MK-801. ICw were determined using a logit-log transformation of the binding data. [3H]Glycine Binding Assay. [3H]Glycine binding to the NMDA receptor associated strychnine-insensitiverecognition site was performed as previously describedam Registry No. 7, 1067-87-4; (2)-8, 129920-47-4; (E)-8, 129920-48-5; (2)-9, 129920-49-6; (E)-9, 129920-50-9; loa, 129920-51-0;lob, 129920-52-1; lla, 129920-53-2; l l b , 129920-54-3; 12a (diastereomer l ) , 129920-55-4; 12a (diastereomer 2), 13000824-1;12b (diastereomer l), 130008-31-0; 12b (diastereomer 2), 130008-32-1;13a (diastereomer l),129920-81-6; 13a (diastereomer f ) , 130008-33-2; 13b (diastereomer l),130008-34-3;13b (diastereomer 2), 130008-35-4; 14, 15916-48-0; 15, 129920-56-5; 16,129920-57-6;17,129920-58-7;18,129920-548; 19,129920-84-9; 20,129920-60-1;21,129920-61-2;22,129920-62-3;23,129920-85-0; (30) Monahan, J. R.; Corpus, V. M.; Hood, W. F.; Thomas, J. W.; Compton, R. P. J. Neurochem. 1987,53, 370-375.
24, 127515-28-0; ( 0 2 5 (diastereomer l), 129920-87-2; (23-25 (diastereomer 2), 130008-45-6;(E)-25 (diastereomer l),13000846-7; (E)-25, 13000847-8; (2)-26 (diastereomer l), 129920-86-1; (2)-26 (diastereomer 2), 130008-42-3; (E)-26 (diastereomer l), 130008-43-4;(E)-26 (diastereomer 2), 13000844-5;27a, 5162-44-7; 27c, 1119-51-3;28a, 129920-63-4;28b, 129920-64-5;28c, 12992065-6; 28d, 129920-66-7;29a, 129920-67-8;29b, 129920-68-9;29c, 129920-69-0;29d, 129920-70-3;30a, 129920-71-4;30b, 129920-72-5; 30c, 129920-73-6; 30d, 129920-74-7; 31a, 129920-75-8; 31b, 129920-76-9;31c, 129920-77-0;31d, 129920-781;32a (diastereomer l), 129920-79-2;32a (diastereomer 2), 130008-25-2;32b (diastereomer l), 130008-26-3;32b (diastereomer 2), 130008-27-4;32c (diastereomer l),129920-80-5;32c (diastereomer 2), 130008-28-5; 32d (diastereomer l), 130008-29-6; 32d (diastereomer 2), 130008-30-9;33a (diastereomer l), 129920-82-7;33a (diastereomer 2), 130008-36-5;33b (diastereomer l),130008-37-6;33b (diastereomer 2), 130008-38-7;33c (diastereomer l),129920-83-8;33c (diastereomer Z),130008-39-8;33d (diastereomer l),130008-40-1; 33d (diastereomer 2), 130008-41-2;NMDA, 6384-92-5;BrCH2CH=CH2, 106-95-6; N2=CHCOOEt,623-73-4;N2=C(COOMe)2, 6773-29-1;N2=CHP03Me2, 27491-70-9.
Synthesis, Antibacterial Activities, and Pharmacological Properties of Enantiomers of Temafloxacin Hydrochloride' Daniel T. W. Chu,* Carl W. Nordeen, Dwight J. Hardy, Robert N. Swanson, William J. Giardina, Andre G. Pernet, and Jacob J. Plattner Anti-infective Research Division, Abbott Laboratories, Abbott Park, Illinois 60064-3500. Received June 6, 1990
Temafloxacin hydrochloride [(f)-7-(3-methylpiperazin-l-yl)-6-fluoro-l-(2,4-difluorophenyl)-l,4-dihydro-4-oxoquinoline-3-carboxylicacid hydrochloride]is a potent member of the 4-pyridone-3-carboxylicacid clans of antibacterial agents and is currently under clinical development as a broad-spectrum antimicrobial agent. It is a racemate having a chiral center at the C3 of the 7-piperazin-1-yl group. The two enantiomers were synthesized and tested for their antibacterial activities. Although no difference in in vitro antibacterial activities was observed, a minor difference in in vivo antibacterial activities was observed. However, they both exhibited similar pharmacological profiles.
In recent years, many clinically important antibacterial agents (such as ciprofloxscin (1)2 and norfloxacin (2)3) having the l-substituted-1,4-dihydro-4-oxopyridine-3carboxylic acid moiety and collectively known as quinolones have been dis~overed.~These agents have been shown to inhibit the topoisomerase enzyme DNA gyrase.6*6 Hence, it may be expected that chirality in the quinolone molecule can have a great impact on the biological activity. Nearly all clinically useful quinolones developed to date, however, are either achiral or racemic mixtures. The S enantiomers of ofloxacin (3)'" and S-25930 (4)g have reA preliminary account of this work has been presented at the 29th ICAAC, Sept 17-20, Houston (Chu, D. T. W.; Nordeen, C. W.; Maleczka, R. E.; Hardy, D. J.; Pernet, A,; Clement, J. J.; Plattner, J. JJ, 1989: Abstract No. 1245. Wise, R.; Andrew, J.; Edward, E. Antimicrob. Agents Chemother. 1983,23, 559. Koga, H.; Itoh, A.; Murayama, S.; Suzue, S.; Irikura, T. J. Med. Chem. 1980,23, 1358. For a recent review, see: Chu, D. T. W.; Fernandes, P. B. Antimicrob. Agents Chemother. 1989, 33, 131. Cornett, J. B.; Wentland, M. P. Annu. Rep. Med. Chem. 1986, 21, 139 and references therein. (a) Fernandes, P. B.; Chu, D. T. W. Annu. Rep. Med. Chem. 1987, 22, 117 and references therein. (b) Fernandes, P. B.; Chu, D. T. W. Ibid. 1988,23, 133 and references therein. Imamura, M.; Shibamura,S.; Hayakawa, I.; Osada, Y.Antimicrob. Agents Chemother. 1986, 29, 163. Mitscher, L. A.; Sharma, P. N.; Shen, L. L.; Chu, D. T. W. J. Med. Chem. 1987, 30, 2283. Gerster, J. F.; Rohlfing, S. R.; Pecore, S.E.; Winandy, R. M.; Stern, R. M.; Landmesser,J. E.; Olsen, R. A,; Gleason, W. B. J. Med. Chem. 1987, 30,839.
cently been reported to possess greater biological activities (10-100-fold) than their antipodes. The R enantiomer of 7-(2-substituted-pyrrolidin-l-yl)quinolone derivative 5a possesses 10-60-fold greater potency than the S enantiomer.1° Minor differences in activity are observed with the two enantiomers of the 7-(3-aminopyrrolin-l-yl)naphthyridine derivative 5b." The enantiomers of the 3-[(ethylamino)methyl]pyrrolidin-1-yl derivative 6, however, were reported to have similar biological activity.12 Temafloxacin hydrochloride (7) [ (i)-7-(3-methylpiperazin-l-yl)-6-fluoro-l-(2,4-difluorophenyl)-1,4-dihydro-4-oxoquinoline-3-carboxylic acid hydrochloride] is a potent quinolone antibacterial agent. It is currently under clinical development and a NDA has been filed in the U.S. It possesses excellent activity against both Gram-positive and Gram-negative bacteria.12 Temafloxacin is a racemate having a chiral center at C-3 of the 7-pipermin-1-ylgroup. Because of the excellent biological activity of 7 and the presence of a chiral center, both enantiomers were synthesized to evaluate their potential differences in biological, pharmacological, and toxicological properties. In this paper, we now report the synthesis and properties of the enantiomers of temafloxacin hydrochloride. (10) Cooper, C. S.; Chu, D. T. W.; Fernandes, P. B.; Pihuleac, E.; Pernet. A. 25th ICAAC.. Seot . 29-0ct 2. Minneaoolis. 1985: -
I
Abstract No. 130. (11) Rosen, T.; Chu, D. T. W.; Lico, I. M.; Fernandes, P. B.; Shen, L.; Borodkin, S.: Pernet, A. G. J . Med. Chem. 1988.31, 1586. (12) Culberton, T. P.; Domagala, J. M.; Nichols, J. B.; Priebe, S.; Skeean, R. W. J. Med. Chem. 1988,31, 503.
0022-262319111834-0168$02.50/0 0 1991 American Chemical Society
Journal of Medicinal Chemistry, 1991, Vol. 34, No. 1 169
Enantiomers of Temafloracin Hydrochloride
Scheme I
F
U C H ,
5a X=CH, R=CHzOH, Rl=R2=H
4
5b X:N,
R:H,
Rl=NH2, R2:F
F&cmH
.& b'
CYNHCzHa
6
7
i 16
a,b
F
Chemistry a x. H , Y.CH, The general method used for the preparation of N9 F arylfluoroquinolones involving an intramolecular nucleok? XzCHj, Y= H philic displacement cyclization reaction has been previously described.14 However, the displacement of the a) CH3COCI/AICl3 b) NaOCIINaOH; HCI c) SOCI,; CH,(COOC,H,)COOH/ known key intermediate 6-fluoro-7-chloro-l-(2,4-difluoro- n-BuLi; HCI d) CH(OC2H5)3/A~20; o,p-difluoroaniline e) NaH/THF phenyl)-l,4-dihydro-4-oxoquinoline-3-carboxylic acid (8) by a substituted amine provided the 7-aminoquinolone Scheme I1 derivative in low yield. In order to improve the synthetic H H yield, we chose a modified synthetic route utilizing 6,7di f luoro- 1- ( 2,4-difluorop hen y 1)- 1,4-dih y d r 0-4-oxoC,H,CH,0CONH 0 ~ N ~: C o o C H 3 a quinoline-3-carboxylic acid (9) as the key intermediate.15 17 L J The C-7 fluorine atom in 9 was found to be a much better leaving group than the C-7 chlorine atom in 8. Hence, the side reaction of the displacement of the fluorine atom in the 2,bdifluorophenyl group as well as the C-6 fluorine group by the amine in 9 was avoided.
-l
-
F-JyJcmH dCWH
b'
I '2'5
23
F
Friedel-Crafh acylation of 1,3,4-trifluorobenzene with acetyl chloride in the presence of aluminum chloride yielded 2,4,5-trifluoroacetophenone(10) (bp 69 OC/8 mmHg; 75%). Oxidation of 10 wit11 5.25% sodium hy(13) Hardy, D. J.; Swanson, R. N.; Hensey, D. N.; Ramer, N. R.; Bower, R. R.; Hanson, C. W.; Chu, D. T. W.; Fernandes, P. B. Antimicrob. Agents Chemother. 1987,31, 1768. (14) Chu, D. T. W.; Fernandes, P. B.; Claiborne, A. K.; Pihuleac, E.; Nordeen, C. W.; Maleczka, R. E.; Pernet, A. G. J. Med. Chem. 1985,28, 1558. (15) The synthesis of racemate temafloxacin utilizing compound 9
has been presented at the 26th ICAAC, Sept 28, New Orleans, 1986 (Chu, D. T. W.; Fernandes, P. B.; Claiborne, A. K.; Maleczka, R. E.; Klock, p.; Shen, L.; Patel, J.; Pernet, A.). An independent synthesis of temafloxacin utilizing similar intermediates was also reported (Narita, H.: Konishi, Y.: Nitta, J.: Nagaski, H.; Kobayihi, Y.;Watanabe, Y. J. Pharm. Sci. Jpn. 1986, 106, 795).
q H
8 R=CI 9 R=F
o < -No H'
b
~
. 2 HCI
H
22
15b
a) H2; Pd/C b) BH3.THF
pochlorite and sodium hydroxide solution gave the 2,4,5trifluorobenzoic acid (11) (mp 96-98 "C; 94%). The acid 11 was converted into the acid chloride upon treatment with thionyl chloride. It was then displaced by the lithium monoethyl malonate dianion to give the @-ketoester 12. Reaction of this 12 with triethyl orthoformate in acetic anhydride yielded an enol ether intermediate, which upon evaporation of solvent to dryness was allowed to react with a slight excess of an 2,4-difluoroaniline in methylene chloride at room temperature to give the enamino keto ester 13 (mp 85-87 "C; 61%). Cyclization of 13 with sodium hydride yielded ethyl 1-(2,4-difluoropheny1)-6,7-difluoro-1,4-dihydro-4-oxoquinoline-3-carboxylate (14) (mp
Chu et al.
170 Journal of Medicinal Chemistry, 1991, Vol. 34, No.I Table I. In Vitro Antibacterial Activities of Temafloxacin (Tema) and Ita Enantiomers
Staphylococcus aureus ATCC 6538P Staphylococcus aureus A5177 Staphylococcus aureus 45 Staphylococcus aurc'us 45 RAR2 Staphylococcus aureus 642A Staphylococcus aureus NCTC 10649 Staphylococcus aureus CMX 553 Staphylococcus epidermidis 3519 Micrococcus luteus ATCC 9341 Micrococcus luteus ATCC 4698 Enterococcus faecium ATCC 8043 Streptococcus bouis A5169 Streptococcus agalactiae CMX 508 Streptococcus pyogenes EESCl Streptococcus pyogenes 930 Const Streptococcus pyogenes 2548 INDUC Escherichia coli Juhl Escherichia coli SS Escherichia coli DC-2 Escherichia coli H560 Escherichia coli KNK437 Enterobacter aerogenes ATCC13048 Klebsiella pneumoniae ATCC 8045 Prouidencia stuartii CMX 640 Pseudomoms aeruginosa BMHlO Pseudomonas aeruginosa A5007 Pseudomonas aeruginosa K799/ WT Pseudomonas aeruginosa K799/61 Pseudomonas cepacia 2961 Acinetobacter SD. CMX 669
0.1 0.2 0.2 0.39 0.2 0.1 0.2 0.2 3.1 1.56 0.78 1.56 0.78 0.39 0.39 0.2 0.05 0.002 0.78 0.02 0.39 0.1
0.1 0.2 0.2 0.39 0.2 0.1 0.2 0.2 3.1 1.56 0.78 1.56 0.78 0.39 0.39 0.2 0.05 0.01 0.78 0.05 0.39 0.1
A
0.1 0.2 0.2 0.39 0.2 0.2 3.1 1.56 0.78 1.56 0.78 0.39 0.78 0.39 0.05 0.005 0.39 0.05 0.39 0.1 0.05 3.1 0.39 0.39 0.39 0.05 12.5 0.1
Figure 1. Serum concentrations of temafloxacin and its enantiomers following a single oral administration of 25 mg/kg in mice.
Biological Results and Discussion Microbiology. The comparative in vitro antibacterial activity of the two enantiomers of temafloxacin (Tema) against representatives of Gram-positive and Gram-negative bacteria is shown in Table I. Data for racemic ter. dfloxacin is provided for comparison. The in vitro antibacterial activities are reported as minimum inhibitory 0.1 0.1 concentration (MIC) in pg/mL. The MIC's were deter6.2 3.1 mined by the 2-fold agar dilution in brain-heart infusion 0.2 0.39 agar. It can be seen from Table I that temafloxacin and 0.39 0.78 0.39 0.39 its R and S enantiomers possess essentially identical an0.05 0.05 tibacterial potency in vitro. The magnitude of the dif6.2 12.5 ferences (3 out of 30 organisms) is within the margin of 0.1 0.1 experimental error. In order to determine the inherent activity, the enan204-205 "C; 77%). Hydrolysis of 14 yielded the key intermediate 6,7-difluoro-1-(2,4-difluorophenyl)-1,4-di- tiomers were assayed" for supercoiling inhibition activity with DNA gyrase isolated from Escherichia coli H560. hydro-4-oxoquinoline-3-carboxylic acid (9). Displacement The ICw values in pg/mL for the 16a (S enantiomer) and of 9 with the respective optically pure 2-methylpiperazine 16b ( R enantiomer) are 0.52 and 0.76, respectively. Within dihydrochlorides (15a or 15b) in pyridine yielded the dethe experimental error, the two enantiomers possess similar sired (S)-(-)-temafloxacin hydrochloride (16a) (mp >300 activity a t the enzymatic level. "C; 49%) and (R)-(f)-temafloxacinhydrochloride (16b) The in vivo potency of the R and S enantiomers as well (mp >300 "C; 42%) (Scheme I). as the racemate (temafloxacin) determined by the mouse The optically pure (S)-2-methylpiperazine was prepared protection test is shown in Table 11. The potency is given earlier14in an extremely low yield by sublimation of glyin EDbovalues which are expressed as the total dose of cyl-L-alaninefollowed by reduction of the diketopiperazine compound in mg/kg required to protect 50% the mice with sodium dimethoxyethoxydihydroalwninate. Because challenged intraperitoneally with organism indicated. The of the very low yield associated with this reduction step, mice were treated subcutaneously (sc) or orally (PO) with an optical resolution was used as a practical synthesis of a specific amount of the test compound divided equally 2-methylpiperazine enantiomers, but only partial resoluto be administered at 1and 5 h after infection. When the tion was achieved.16 The requisite optically pure enan95% confidence limits are taken int3 consideration, the tiomers were prepared in good yield, however, by a moddata showed that in vivo, following both oral or subcutaified route as outlined in Scheme 11. Deprotection of neous administration to the mice, both (S)-(-)-temafloxcarbobenzoxyglycyl-L-alaninemethyl ester (17) by hydroacin (16a) and (R)-(+)-temafloxacin (16b) possess comgenolysis yielded glycyl-L-alanine methyl ester (18) inparable potency against Staphylococcus aureus NCTC termediate, which, without isolation, was allowed to cyclize 10649,Pseudomonas aeruginosa A5007, and Escherichia to give the diketopiperazine 19 (mp 246-247 "C; 83%). coli Juhl. Against Streptococcus pneumoniae 6303, the Reduction of 19 (S enantiomer) with borane-tetrahydroS-(-)-enantiomer is slightly more active than the R-(+)furan complex yielded the (S)-(-)-2-methylpiperazinedienantiomer. (*)-Temafloxacin possesses intermediate hydrochloride (15a) (mp >300 "C;53%). By use of a potency. It is possible that the slightly higher in vivo similar synthetic sequence, the (R)-(+)-2-methylpiperazine potency of (S)-(-)-temafloxacin is the result of better dihydrochloride (15b) was prepared from carbobenzoxypharmacokinetics and metabolism in the animal model, glycyl-D-alanine methyl ester. By use of the above synsince all the test compounds are equally potent in vitro. thetic route, racemization is normally not expected to The plasma concentrations of (R)-(+)-and (S)-(-)-teoccur. The specific rotations for these two enantiomers mafloxacin after an oral dose of 25 mg/kg of the test = -2.73 (H20)for 15a and [a]26D = +2.63 (H20) compounds administered to mice is given in Figure 1. The for 15b), however, showed a slight difference. Although S-(-) enantiomer provided an area under the serum curve this difference may be the result of experimental error, a (AUC) of 9.4 mcg/hr/mL while the R-(+) enantiomer gave slight partial racemization during their syntheses may not an AUC of 4.7 pg h 1 mL-'. The plasma concentration of be totally excluded. (16) Armarego, W. L. F.; Schou, H.; Waring, P,J. Chem. Res. Synop. 1980, 133.
(17) Hogberg, T.;Khanna, I.; Drake, S. D.; Mitscher, L. A.; Shen, L. L. J . Med. Chem. 1984,27, 306.
Enantiomers of Temafloxacin Hydrochloride
Journal of Medicinal Chemistry, 1991, Vol. 34, No. 1 171
Table 11. Mouse Protection Test of Temafloxacin (Tema) and Its Enantiomers ED,, mg/kg (95% confidence limits) compound" route test 1 test 2 S(-)-Tema sc 0.9 (0.6-1.4) 1.5 (1.0-2.2) I?(+)-Tema sc 2.0 (1.3-3.0) 2.8 (1.8-4.2) Tema sc 1.5 (1.0-2.2) 1.3 (0.8-2.2) S(-)-Tema PO 3.1 (1.8-5.3) 3.6 (2.1-6.3) R(+)-Tema PO 4.4 (2.5-7.7) 6.8 (4.3-10.7) Tema PO 6.0 (3.8-9.5) 3.2 (1.6-6.3) S. pneumoniae 6303 (100xLD,) S(-)-Tema SC 4.7 (3.0-7.6) R(+)-Tema sc 17.2 (11.3-26.3) Tema sc 6.2 (3.9-9.9) S(-)-Tema PO 5.9 (3.8-9.1) R(+)-Tema PO 19.5 (11.6-33.0) Tema PO 10.2 (6.4-16.4) E. coli Juhl (lOOXLD,) S(-)-Tema sc 0.3 (0.2-0.5) 0.5 (0.3-0.8) 1.2 (0.7-2.1) 0.9 (0.5-1.8) R(+)-Tema sc Tema sc 0.5 (0.3-0.8) 0.6 (0.4-1.0) S(-)-Tema PO 2.3 (1.7-3.1) 2.5 (1.9-3.2) R(+)-Tema PO 3.6 (1.8-7.5) 4.5 (2.3-8.9) Tema PO 3.7 (2.1-6.5) 5.0 (3.2-7.9) P.aeruginosa A5007 (lOOxLD,) S(-)-Tema sc 10.3 (6.6-16.3) 10.5 (5.7-19.2) R(+)-Tema sc 10.7 (6.6-17.2) 16.5 (10.5-25.8)b Tema sc 10.3 (6.6-16.3) 12.5 (7.9-19.8) S(-)-Tema PO 19.9 (12.8-31.1) 25.0 (15.8-39.6) R(+)-Tema PO 62.0 (31.3-122.9) 51.3 (30.7-85.6)' Tema PO 26.6 (13.7-51.4) 26.6 (13.7-51.4) a Each comDound was tested at three dose with 10 animals Der dose. EDw is the median dose calculated from cumulative mortalities. Infecting dose 1000XLD,. test organism dose S. aureus NCTC 10649 (100xLD,)
Table 111. Comparative Physical Properties of Temafloxacin and Its Enantiomers water solubility water solubility" compound in water, mg/mL at pH 7.5, mg/mL log pb -0.27 S enantiomer 5.5 0.37 5.9 0.36 -0.27 R enantiomer 6.5 0.67 -0.29 temafloxacin Water solubility of test compound in 0.05 M pH 7.5 phosphate buffer. bThe partition coefficients in octanol/water at pH 7.5 buffer.
Figure 2. Serum concentrations of temafloxacin and its enantiomers following a single subcutaneous administration of 25 mg/kg in mice.
R and S enantiomers at a single subcutaneous dose of 25 mg/kg to mice is shown in Figure 2. The AUCs for S and R enantiomers were 11.2 and 5.7 pg h-' mL-l, respectively. As indicated in Figures 1 and 2, the S-(-)enantiomer had almost 2-fold greater area under the serum curve than the
R-(+) enantiomer. Although the peak serum level for the
enantiomer two enantiomers are almost identical, the S-(-) persisted at a higher concentration,up to 8 h after dosing. The differences may account for the slight difference in in vivo efficacy in the mouse protection tests. Solubility Studies. The slight difference in pharmacokinetic properties with the two enantiomers may be due to their differences in physical properties. The solubilities and the partition coefficients in octanol/water (log P)of (f)-temafloxacin and its enantiomers were determined as shown in Table 111. However, both enantiomers exhibit essentially identical solubility and log P values. It should be noted that racemic temafloxacin has twice the solubility value as its enantiomers a t physiological pH which may be of practical importance. Pharmacology. Central nervous system (CNS) stimulation is a characteristic side effect of some members of quinolone antibacterials currently in clinical use. Mild headache, dizziness, sleep disturbance, or mood alterations are the common side effects. Seizures have been reported
in small numbers of patients receiving ciprofloxacin18and enoxacin.lg Amfonelic acid (23),a quinolone derivative, produced a marked CNS stimulant effect on locomotor activity.20 With the availability of both temafloxacin enantiomers, the potential CNS effects of (R)-and (S)temafloxacins were evaluated in male Sprague-Dawley rats by a measurement of locomotor activity. The effects of (R)-and (S)-temafloxacin and oxolinic acid on the spontaneous motor activity of rats are shown in Table IV. The enantiomers were administered at doses of 30,100, and 300 mg/kg, intraperitoneally (ip), and oxolinic acid was administered at doses of 30,100, and 300 mg/kg, orally (PO). A t 30 min after the administration of the test compounds or a volume dose of the vehicle (methylcellulose), the rats were placed into individual circular activity chambers. Spontaneous locomotor activity measurements were derived from the interruption of photocell beams which are strategically placed within the chambers. Activity counts were recorded at 15 and 60 min after the start of the test. The treatments were administered on a random basis. At intraperitoneal doses at 30 and 100 mg/kg, no significant effects were seen with either enantiomer of temafloxacin. However, at an extremely high ip dose of 300 mg/kg, both enantiomers significantly reduced locomotor activity a t ~
~~
~~
~
(18) hoof, S.; Wollschlager, C.; Klan, F. J. Antimicrob. Chemother. 1986, 18 (Suppl. D), 139. (19) Davies, B. I.; Maesen, F. P. V.; Teengs, J. P. J. Antimicrob. Chemother. 1984, 14 (Suppl. C),83. (20) Aceto, M. D.; Harris, L. S.; Lesher, G. V.; Brown, T. G., Jr. J. Pharmacol. Exp. Ther. 1967, 258, 286.
172 Journal of Medicinal Chemistry, 1991, Vol. 34, No. 1
Table IV. Effects of Intraperitoneal Administration of (R)-and (S)-Temafloxacinon the Spontaneous Locomotor Activity of Rats mean activity counts f SEM dose, treatment mg/kg route 0-15 min 0-60min 736 f 46 vehicle 962 f 59 699 f 57 (R)-temafloxacin 863 f 92 30 822 f 92 673 f 90 100 291 f 75' 300 183 f 70' 571 f 70 vehicle 922 f 115 549 f 52 751 f 99 30 (S)-temafloxacin 100 386 f 64 763 f 97 270 f 47' 354 f 51" 300 1 methamphetamine 1462 f 118" 3728 f 478' chlorpromazine 31 f 28O 68 f 37' 15 vehicle 819 f 55 1087 f 106 oxolinic acid 30 972 f 65 1746 f 165" 1074 f 79 100 2561 f 4541' 1031 f 87 300 3085 f 391" "Denotes that the group ( N = 6 with the exception of oxolinic acid where N = 12) is significantly different from vehicle at 0.05 level of confidence using one-way analysis of variance and the Newman-Keuls procedure*' on transformed data.
both the 15- and 60-min measurements. In general, both compounds had similar effects on t h e spontaneous locomotor activity of rats. I n contrast, oxolinic acid at 30, 100, a n d 300 mg/kg, PO produced significant increase in locomotor activity as shown in Table IV. T h e effects of methamphetamine (1mg/kg, ip), a known CNS stimulant, and chlorpromazine (15 mg/kg, ip), a known CNS depressant on locomotor activity was also included for comparison. Neither enantiomers of temafloxacin showed the marked stimulant or depressant activity associated with these centrally active drugs. Summary of Results. The enantiomers of temafloxacin (7) have been synthesized. T h e y were evaluated in vitro against both Gram-positive a n d Gram-negative bacteria. Their in vivo efficacy, pharmacokinetic properties, and CNS pharmacology were assessed. (*)-Temafloxacin was shown t o be as active as its R-(+) or S-(-) enantiomers in vitro. A slightly better pharmacokinetic property was observed for (SI-(-)-temafloxacin in mice. It remains to be seen whether other animal species will provide similar differences. Because the enantiomers have identical antibacterial potency in vitro, as well as similar CNS activity on spontaneous locomotor activity in rats, and because the racemate has higher water solubility, t h e continuation of clinical development for t h e racemic temafloxacin remains appropriate.
Experimental Section Unless otherwise noted, materials were obtained from commercial suppliers and used without further purification. Melting points were taken in a Thomas-Hoover capillary apparatus and was uncorrected. NMR spectra were determined on a General Electric GN-300 spectrometer operating at 300.1 MHz. Chemical shifts are expressed in ppm downfield from internal tetramethylsilane. Significant 'H NMR data are tabulated in the order: number of protons, multiplicity (8,singlet; d, doublet, t, triplet, q, quartet; m, multiplet; b, broad), coupling constant(s) and designation. The IR spectra were recorded on a Perkin-Elmer Model 710A infrared spectrometer. Mass spectra were obtained with a Hewlett-Packard 5985A mass spectrometer or a Kratos MS-50 instrument with El source (70 eV). The IR, NMR and mass spectra data of all compounds were consistent with the assigned structures. Elemental analyses were obtained for all new compounds reported. Carbon, hydrogen, and nitrogen analyses (unless otherwise specified) were within f0.4% of the theoretical values. Solutions were dried over magnesium sulfate. E. Merck silica gel (230-400 mesh) obtained from VWR Scientific was used for column chromatography, and yields of the reactions were not
Chu et al. optimized. Elemental analyses were performed by the Abbott analytical department and IR, NMR, and mass spectra were recorded by the Abbott structural chemistry department. 2,4,5-Trifluoroacetophenone(10). Acetyl chloride (10.67 mL, 0.15 mol) was added dropwise to aluminum chloride (29.33 g, 0.22 mol) over a 15-min period. The reaction was exothermic with evolution of gas observed. The mixture was heated at 50 "C for 1 h. 1,2,4-Trifluorobenzene (13.2 g, 0.1 mol) was then added to the mixture over a 5-min period. The mixture was heated at 80 "C for 22 h. The mixture was cooled to room temperature and was poured into a mixture of 200 g of ice and 25 mL of concentrated hydrochloric acid mixture. After 0.5 h, it was extracted several times with methylene chloride. The combined methylene chloride extract was washed several times with dilute hydrochloric acid and then water. The solution was dried, filtered, and distilled at 69 "C (8 mmHg) to give 13 g of a mobile liquid (75% yield) C, H. NMR (CDC13): 6 2.67 (3 H, d, JH-F of 10. Anal. (CBH5F3) = 6 Hz, CH3),7.06 (1 H, m, aromatic H), 7.78 (1H, m, aromatic HI. 2,4,5-TrifluorobenzoicAcid (I 1). Sodium hydroxide (42 g, 1.05 mol) was dissolved in 100 mL of water and was added to 2500 mL of commercial bleach (sodium hypochlorite, 5.25%) with rapid stirring. 2,4,5-Trifluoroacetophenone (91.5g, 0.526 mol) was then added over a 25-min period. The reaction temperature was maintained below 25 "C. After 22 h, excess sodium bisulfite solution was added to decompose the excess of sodium hypochlorite until KI test paper was negative. The mixture was adjusted to pH 2 by the addition of concentrated hydrochloric acid. The precipitate was filtered and washed with water (2 X 200 mL) and dried. The aqueous phase was extracted several times with methylene chloride, dried, and evaporated to dryness to give a solid. The combined solid gave 86.73 g (94%) of 2,4,5-trifluorobenzoic acid (ll),mp 96-98 "C. Anal. (C7H3F302) C, H. NMR (DMSO-d,): 6 7.71 (1 H, m, aromatic H), 7.91 (1 H, m, aromatic H), 13.67 (1 H, bs, COOH). Ethyl (2,4,5-Trifluorobenzoyl)acetate(12). After a mixture of 2,4,5-trifluorobenzoic acid (9.65 g, 54.8 mmol) and thionyl chloride (45 mL) was heated at 80 "C for 4 h, the solvent was removed by evaporation under reduced pressure, yielding a mobile oil (2,4,5-trifluorobenzoylchloride). Monoethyl malonate (17 g, 128.8 mmol) and 5 mg of biquinoline were dissolved in 350 mL of dry tetrahydrofuran (THF), and the solution was cooled to -30 "C. A solution of approximately 2.6 M n-butyllithium in hexane was added until a pink color remained at -5 "C (99 mL). The suspension was then cooled to -50 "C. The acid chloride, obtained as described above, was dissolved in 70 mL of THF. This was added to the suspension dropwise. After 0.5 h, the dry ice bath was removed and the reaction mixture was allowed to warm up to room temperature. The reaction mixture was acidified with 250 mL of 1 N hydrochloric acid and was extracted with ether. The ether solution was dried and filtered. Evaporation of the solvent to dryness yielded an oil. This was purified by use of Kugelrohr distillation to give 13.2 g (98%) of 12. NMR (CDC13): 6 (two sets of signals), 1.22 (1.8 H, t, J = 7 Hz, ethyl CHJ, 1.27 (1.2 H, t, J = 7 Hz, ethyl CH,), 3.30 (0.8 H, s, CH2),4.14 (1.2 H, q, J = 7 Hz, ethyl CH2),4.19 (0.8 H, q, J = 7 Hz, ethyl CH2),5.76 (0.6 H, s, vinyl H), 6.91 (1 H, m, aromatic H), 7.66 (1 H, m, aromatic H), 12.65 (0.6 H, s, enol OH). Ethyl 3-(2,4-Difluoroanilino)-2-(2,4,5-trifluorobenzoyl)acrylate (13). A solution of 12 (8.4 g, 34.1 mmol) in triethyl orthoformate (85 mL, 51.2 mmol) and acetic anhydride (21.5 mL, 153 mmol) was heated at 125 "C for 2 h with removal of the ethyl acetate formed during the reaction. The solution was evaporated under reduced pressure to a mobile oil that was dissolved in methylene chloride (100 mL). 2,4-Difluoroaniline(3.82 mL, 37.5 mmol) was added to the solution. After 45 min, the solution was evaporated to dryness and crystallized from 10% ether in hexane solution, yielding 8 g (61%) of 13, mp 85-87 "C. Anal. (C18HI2F5NO3)C, H, N. NMR (CDCI,): 6 1.03 (3 H, t, J = 7 Hz, ethyl CH,), 4.13 (2 H, q, J = 7 Hz, ethyl CH2),7.06 (5 H, m, aromatic H), 8.45 (1H, d, J = 12 Hz, olefinic H), 12.43 (1 H, bd, J = 12 Hz, NH). Ethyl 1-(2,4-Difluorophenyl)-6,7-difluoro-1,4-dihydro-4oxoquinoline-3-carboxylate(14). A 60% sodium hydride in oil suspension (730 mg, 18.3 mmol) was slowly added to a cold solution of 13 (6.85 g, 17.8 mmol) in tetrahydrofuran (100 mL).
Enantiomers of Temafloxacin Hydrochloride The mixture was heated at 50 OC for 13 h under nitrogen atmosphere and was cooled and acetic acid (1mL) was added. The solution was evaporated under reduced pressure to dryness. The residue was dissolved in methylene chloride (200 mL). The solution was washed with saturated sodium chloride solution and the organic portion was separated and dried. Upon evaporation to dryness, the residue was crystallized from ether and filtered, yielding 5 g (77%) of 14, mp 204-205 "C. Anal. (C18HllF4N03) C, H, N. NMR (CDC13): 6 1.30 (3 H, t, J = 7 Hz, ethyl CH3), 4.37 (2 H, d, J = 7 Hz, ethyl CH2),6.73 (1 H, m, aromatic H), 7.25 (2 H, m, aromatic H), 7.70 (1 H, m, aromatic H), 8.17 (1H, m, aromatic H), 8.02 (1 H, s, olefinic H). 1-(2,4-Difluorop henyl)-6,7-difluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic Acid (9). Hydrochloric acid (6 N, 25 mL) was added to a solution of 14 (4 g, 10.96 mmol) in trifluoroacetic acid (25 mL). The mixture was heated at 70 OC for 2 days. It was cooled, 200 mL of water was added, and the mixture was filtered. The solid was washed with water and dried, yielding 3.62 g (98%)of 9, mp 240 "C. Anal. (Cl&I7F4NO3)C, H, N. NMR (DMSO-d6) 6 7.44 (2 H, m, aromatic H), 7.73 (1H, m, aromatic H), 7.91 (1H, m, aromatic H), 8.36 (1 H, m, cromatic H), 8.98 (1 H, s, olefinic H), 14.45 (1 H, bs, OH). (S)-3-Methyl-2,5-dioxopiperazine(19). A solution of carbobenzyloxyglycinesuccinimido ester (134 g, 430 mmol) in THF (600 mL) was added dropwise to a solution of L-alanine methyl ester hydrochloride (67.19 g, 480 mmol) and sodium bicarbonate (121 g, 1.44 mol) in water (600 mL) at 10 "C. After the addition, the reaction was allowed to warm up to room temperature and was stirred for 16 h. The solvent was removed under reduced pressure and the residue was taken up in ethyl acetate (2.5 L) and washed twice with water. The organic portion was separated and dried. Removal of the solvent yielded 114.1 g (90%) of 17, mp 91-93 OC. A solution of 17 obtained from above in 1 L of methanol was hydrogenated under hydrogen atmosphere at 4 atm pressure with 10% palladium on carbon for 18 h. The solution was then filtered and the residue was washed in 3 L of methanol. The combined solution was filtered through a 0.45-pm nylon filter to remove the residual catalyst. Concentration of the filtrate to dryness yielded a solid which was digested with 1 L of boiling ethanol. It was cooled and filtered, yielding 41.17 g (83%)of 19, mp 246-247 OC. Anal. (C5H8Nz02)C, H, N. NMR (D20): 6 1.47 (3 H, d, J = 6.5 Hz, CH3), 4.05 (2 H, dq, J = 17 Hz, CHJ, 4.16 (1 H, m, CH). [.]25D = -3.78 (H2O). By use of this procedure, reacting D-alanine methyl ester hydrochloride instead of L-alanine methyl ester hydrochloride with carbobenzyloxyglycinesuccinimidoester, (R)-3-methyl-2,5-dioxopiperazine (22) was obtained in 85% yield, mp 247.5-249.5 OC. [.]26D = +3.56 (HzO). Anal. (C5H8N202)C, H, N. NMR (D20): 6 1.47 (3 H, d, J = 6.5 Hz, CH3), 4.06 (2 H, dq, J = 17 Hz, CH2),4.16 (1H, m, CH). (S)-2-Methylpiperazine Dihydrochloride (15a). (S)-3Methyl-2,5-dioxopiperazine (19) (41.17 g, 320 mmol) was suspended in T H F (1 L). A 1.35-L sample of 1 M borane-tetrahydrofuran complex (1.35 mol) was then added dropwise to the above stirring suspension over 1 h. Gas evaluation occurred and the mixture became nearly solution at the end of the addition. The mixture was refluxed for 16 h. It was cooled to room temperature and 200 mL of methanol was added over a 1-h period. Gas evolution occurred and the reaction mixture was concentrated to dryness. The residue was redissolved in methanol (500 mL) and excess rnethanolic HC1 solution was added and the solution was refluxed for 2 h. It was cooled and allowed to stand at room temperature ovemight. The precipitate was filtered to yield 29.61 g (54%) 15a, mp >300 OC. [a]26.6~ = -2.73 (H20). Anal. (C5H12N2.2HC1.0.4H20) C, H, N. NMR (DZO): 6 1.49 (3 H, d, J = 6.5 Hz, CH3),3.27 (1, m, CH), 3.47 (2 H, m, NCH2),3.78 (4 H, m, 2NCH2). By use of this procedure, (R)-3-methyl-2,5-dioxopiperazine (22) gave (R)-2-methylpiperazinedihydrochloride (15b) in 43% yield, ~ +2.63 (H20). Anal. (C5Hl2N2.2HC1)C, mp >300 "C. [ n I z 6 = H, N. NMR (DzO): 6 1.43 (3H, d, J = 6.5 Hz, CHB), 3.24 (1 H, m, CHI, 3.44 (2 H, m, NCH2),3.74 (4 H, m, 2NCHzj. l-(2,4-Difluorophenyl)-6-fluoro-7-( (S) - 3 - m e t h y l Acid piperazin-1-y1)-1,4-dihydro-4-oxoquinoline-3-carboxylic Hydrochloride (16a, (S)-Temafloxacin Hydrochloride). (S)-(-)-2-Methylpiperazinedihydrochloride (19) (25.62 g, 148
Journal of Medicinal Chemistry, 1991, Vol. 34, No. 1 173 mmol) was added to a mixture of 9 (25 g, 74 mmol) and triethylamine (37.44 g, 370 "01) in pyridine (500mL). The reaction mixture was heated at 70 "C with stirring for 18 h. The reaction was cooled to room temperature and concentrated under reduced pressure to dryness. Ethanol was added to digest it at boiling for 30 min. The mixture was cooled and filtered, yielding 19.84 g of a solid. This solid was dissolved in 700 mL of hot dilute hydrochloric acid. A small amount of insoluble impurities was filtered off and the solution was evaporated to dryness. The residue was crystallized from ethanol-water to yield 16.35 g (49%) of 16a, mp >300 OC. Anal. (C21H18F3N303-HCl.H20) C, H, N. [cY]~"~= D -10.33 (HZO). NMR (DMs0-d~):6 1.26 (3 H, d, J = 7 Hz, CH3), 2.95 (1 H, m, NCH), 3.11 (2 H, m, NCH2), 3.48 (4 H, m, 2NCH2),6.41 (1 H, d, JH-F = 7 Hz, aromatic H), 7.47 (1 H, m, aromatic H), 7.70 (1 H, m, aromatic H), 7.95 (1 H, m, aromatic H), 8.04 (1 H, d, JH-F = 12 Hz, aromatic H), 9.54 (1H, bs, NH). By use of this procedure, 1-(2,4-difluoropheny1)-6-fluoro-7((R)-3-methylpiperazin-l-yl)-1,4-dihydro-4-oxoquinoline-3carboxylic acid hydrochloride (16b, (R)-temafloxacinhydrochloride was prepared from (R)-2-methylpiperazine dihydrochloride (22) in 42% yield, mp >300 OC. Anal. (C21HlsF3N303-HCl-H20) C, H, N. [a]27'5~ = +11.33 (HZO). NMR (DMSO-dg): 6 1.26 (3 H, dd, J = 7 Hz, CH,), 2.97 (1 H, m, NCH), 3.11 (2 H, m, NCH2), 3.46 (4 H, m, 2NCH2), 6.40 (1 H, d, JH-F = 7 Hz, aromatic H), 7.46 (1 H, m, aromatic H), 7.73 (1 H, m, aromatic H), 7.95 (1H, m, aromatic H), 8.03 (1H, d. JH-F = 1 2 Hz, aromatic H), 8.85 (1 H, s, NH). In Vitro Antibacterial Activity. The in vitro antibacterial activity of the test compounds was tested in a side-by-side comparison with temafloxacin and determined by conventional agar dilution procedures. The organisms were grown overnight in brain-heart infusion (BHI) broth (Difco 0037-01-6) a t 36 OC. Two-fold dilutions of the stock solution (2000 pg/mL) of the test compound were made in BHI agar to obtain the test concentration ranging from 200 to 0.005 pg/mL. The plate was inoculated with approximately lo4organisms. It was then incubated at 36 OC for 18 h. The minimal inhibitory concentration (MIC) was the lowest concentration of the test compound that yielded no visible growth on the plate. In Vivo Antibacterial Activity. The in vivo antibacterial activity of the test compounds was determined in CF-1 female mice weighing approximately 20 g. Aqueous solutions of the test compounds were made by dissolving the hydrochloride salt in distilled water and diluting it with distilled water to the desired volume. The median lethal dose of the test organism was determined as follows. After 18-h incubation, the cultures of test organism in BHI broth were serially diluted by using 10-folddilutions in 5% (w/v) hog gastric mucin. Cultures (0.5 mL), dilution from lo-' to lo4, were injected intraperitoneally into mice. The LDw for the test organism was calculated from cumulative mortalities on the sixth day by using the Reed and Muench procedure.22 After 18 h culture of the above was diluted in 5% (w/v) hog gastric mucin to obtain 100 times the LDw, and 0.5 mL was injected intraperitoneally into mice. The mice were treated subcutaneously (sc) or orally (PO) with a specific amount of the test compound divided equally to be administered at 1and 5 h after infection. A group of 10 animals each for at least three dose levels was thus treated, and the deaths were recorded daily for 6 days. Ten mice were left untreated as infection control. EDw values were calculated from the cumulative mortalities on the sixth day after infection by using the trimmed version of the logit method.23 Solubility Studies. The solubility studies were run using excess drug on a fixed volume of distilled water or in 0.05 M, pH 7.5 potassium phosphate buffer at ambient temperature. Samples were filtered and the filtrates were assayed by UV. A HewlettPackard 8452 diode array spectrophotometer equipped with a (21) Snedecor, G.W.;Cochran, W. G. Statistical Methods, 6th ed.); The Iowa State University Press: Ames, Iowa, 1967; pp 273-275. (22) Reed, L.J.; Muench, H. Am. J. Hyg.1938, 27, 493. (23) Hamilton, M. A,; RUBSO, R. C.; Thurston, R. V. Enuiron. Sci. Technol. 1977, 11, 714.
J. Med. Chem. 1991, 34, 174-180
174
Chemstation was used for all UV spectrophotometricmeasurements. Complete spectra were quantitatively compared to known standards with use of a large segment of the spectra. DNA Gyrase Inhibitor Activity. The DNA holoenzyme was prepared according to the procedure described2‘ using a heparin-sepharose affinity column. The DNA gyrase supercoiling activity was assayed by a gel electrophoresis technique.% A 1% agarose horizontal gel slab was used. The amount of relaxed plasmid (CoE1) band and the supercoil band formed was de(24) Gellert, M.; Mizuuchi, K.; ODea, M. H.; Nash, H. A. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 3876. (25) Otter, R.;Cozzarelli, M. R. Methods Enzymol. 1983, IOOB, 171.
termined by tracing the photographic negatives of the gel on a LKB Model 2022 Ultroscan densitometer. Because of the noncompetitive nature of the inhibitors, Ki may be determined as the concentration that caused 50% inhibition of the supercoil band formation.
Acknowledgment. We wish t o thank the analytical research departments of Abbott Laboratories for spectral measurement and elemental analysis; Drs.Linus Shen and Saul Borodkin for providing the DNA gyrase inhibitory, water solubility, and log P data; Mr. Robert E. Maleczka, Jr., for his technical assistance and Ms. Cynthia Shepherd-Davis for typing t h e manuscript.
Synthesis and Biological Evaluation of Dipeptidyl and Tripeptidyl Polyoxin and Nikkomycin Analogues as Anticandidal Prodrugs Eduardo Krainer,’ Jeffrey M. Becker,* and Fred Naider*-t Department of Chemistry, College of Staten Island, City University of New York, Staten Island, New York 10301, and Department of Microbiology and Program in Cellular, Molecular, and Developmental Biology, University of Tennessee, Knoxville, Tennessee 37996. Received February 8, 1990 Nine analogues (1-5,9-12) of the peptidyl nucleoside antibiotics nikkomycin and polyoxin were synthesized and tested for their biological properties against different strains of the pathogenic yeast Candida albicans. The tripeptidyl series of analogues (1-5) was designed to behave as prodrugs, releasing a toxic moiety upon enzymatic hydrolysis inside the cell. The dipeptidyl series (9-12) was designed as double-targeted drugs,being themselves toxic and releasing a toxic amino acid upon hydrolysis. All the analogues were prepared by coupling suitably protected amino acid p-nitrophenyl esters to 1-(5’-amino-5’-deoxy-a-~-allofuranuronosy~)uraci~ (UPOC) or the correspondingpolyoxins and nikkomycins, with subsequent removal of the protecting group. Improved coupling yields were observed when DMSO was used as the solvent. Products were purified with use of reversed-phase HPLC and, in one case, diastereomeric products (compound 11) were resolved by using this procedure. One of the tripeptidyl nikkomycins behaved as a prodrug but none of the compounds, as measured by in vitro testing, proved more effective than nikkomycin as an anticandidal agent.
Introduction Opportunistic infections by Candida albicans are major contributors t o morbidity and mortality in immunocomSince t h e drugs currently in use for the promised treatment of candidiasis suffer from significant clinical limitations, a clear need exists for t h e development of effective anticandidal drugs. Polyoxins and nikkomycins, closely related families of peptidyl nucleoside antibiotics, produced by species of Streptomyces, are potent competitive inhibitors of C. albicans chitin ~ y n t h e t a s e . However, ~~~ these compounds are not very effective fungicidal agents, when measuring growth of C.albicans in culture. These findings could be the result of the failure of these antibiotics to accumulate intracellularly or t o their metabolism by t h e yeast. Degradation inside the cell does not seem to be the problem, since polyoxins have been shown t o resist Candida peptidases! In contrast, polyoxin and nikkomycin permeation into the cell takes places through peptide permeases; this is t h e step t h a t appears t o be rate limiting in t h e case of
C. a l b i ~ a n s . ~ * ~ T o explore increasing the uptake of peptidyl nucleosides by C.albicans, we have prepared a number of tripeptidyl nikkomycins and polyoxins using amino acid residues expected to improve recognition by the tripeptide transport system (Table I). These tripeptide prodrugs should not be inhibitors of chitin synthetase but should be hydrolyzed t o toxic dipeptides. We have also synthesized a variety
t
College of Staten Island. University of Tennessee.
of polyoxin analogues containing a known antimetabolite (Table 11, 10-12). These multitargeted drugs have the potential t o inhibit chitin synthetase and release the toxic amino acids oxalysine,’ m-fluorophenylalanine8 and Nj-
(4-methoxyfumaroyl)-~-2,3-diaminopropanoic acid (FMDP)? In this communication we report the synthesis of the above analogues and the evaluation of their stability and anticandidal activity. Chemistry 1-(5’-amino-5’-deoxy-a-~-allofuranuronosyl)uracil, which we have previously designated UPOC (uracil polyoxin C),S is t h e carboxyterminus amino acid of the synthetic dipeptides 9, 10,ll, 12,and of tripeptide5 1 and 2. It was synthesized from uridine, according to Damodaran et al.l0 (1) Armstrong, D. Ann. N. Y.Acad. Sci. 1988, 544, 443. (2) Bodey, G. P. Ann. N. Y. Acad. Sci. 1988,544, 431. (3) Naider, F.;Becker, J. M. Current Topics in Medical Mycology; McGinnis, M., Ed.; Springer-Verlag: New York, 1988, pp 170-198. (4) Krainer, E.; Khare, R. K.; Naider, F.; Becker, J. M. Anal. Biochem. 1987, 160, 233. (5) Shenbagamurthi,P.;Smith, H. A.; Becker, J. M.; Steinfeld, A.; Naider, F. J. Med. Chem. 1983,26, 1518.
(6) Yadan, J.-C.; Gonneau, M.; Sarthou, P.; Le Goffic, F. J.Bacteriol. 1984, 160, 884. (7) Qinzhu, B.; Hailan, Z.; Shaohua, X.;Yulin, H.; Shuxun, Lo; Yunfen, Y. Acta Microbiol. Sin. 1981,21, 218. (8) Kingsbury, W. D.; Boehm, J. C.; Mehta, R. J.; Grappel, S.F. J. Med. Chem. 1983,26, 1725. (9) Andruszkiewicz, R.;Chmara, H.; Milewski, S.;Borowski, E. Int. J. Pept. Protein Res. 1986, 27, 449.
0022-262319111834-0174$02.50/0 0 1991 American Chemical Society
